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Characterization of a Fission Yeast Gene, Gpa2, That Encodes a Subunit Involved in the Monitoring of Nutrition

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Characterization of a Fission Yeast Gene, Gpa2, That Encodes a Subunit Involved in the Monitoring of Nutrition Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Characterization of a fission yeast gene, gpa2, that encodes a subunit involved in the monitoring of nutrition Takako Isshiki, Nobuyoshi Mochizuki, 1 Tatsuya Maeda, 2 and Masayuki Yamamoto s Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Tokyo 113, Japan The Schizosaccharomyces pombe gpa2 gene was cloned by hybridization with a eDNA for Dictyostelium discoideum G,vl. It encodes a homolog of G-protein ot-subunits with 354 amino acids and a predicted molecular mass of 40,522. Disruption of gpa2 slows cell growth but is not lethal. Cells defective in gpa2 mate and sporulate readily in the presence of plentiful nutrition, bypassing the requirement of nitrogen starvation for the initiation of sexual development. These phenotypes mimic those of cells defective in cyrl encoding adenylyl cyclase. The level of cAMP in gpa2 null mutants is only one-third of the wild-type level. Mutations in gpa2 that are likely to inhibit the GTPase activity of the gene product cause a slight increase in intracellular cAMP levels and result in leaky sterility. The cAMP level reaches 20 times as high as the wild-type level if a cell carries both this type of gpa2 mutation and a null mutation in pdel encoding phosphodiesterase. Cells defective in gpa2 fail to produce cAMP in response to glucose stimulation. These results suggest that Gpa2 is involved in the determination of the cAMP level according to nutritional conditions, most likely as a positive regulator of adenylyl cyclase. [Key Words: Schizosaccharomyces pombe; G protein; cAMP production; glucose; sexual differentiation] Received August 6, 1992; revised version accepted October 12, 1992. Cells of the fission yeast Schizosaccharomyces pombe receptors that carry seven membrane-spanning domains. initiate sexual development, namely mating, meiosis, The receptors on the cell membrane bind their specific and sporulation, in response to nitrogen starvation (Leu- ligands, and this signal is transmitted into the cell by the pold 1970; Egel 1973). Nitrogen starvation reduces the G proteins (Gilman 1987; Dohlman et al. 1991). Our pre- intracellular cAMP level by 50% (Fukui et al. 1986; Mo- vious screen identified the S. pombe gpal gene encoding chizuki and Yamamoto 1992). This reduction is a signal an a-subunit of a G protein that we concluded to be for induction of the stell gene, which encodes a tran- coupled with the mating pheromone receptors (Obara et scription factor responsible for the activation of genes al. 1991). We have now identified an S. pombe gene that involved in mating and meiosis (Watanabe et al. 1988; encodes another Get subunit and named it gpa2. Genetic Sugimoto et al. 1991 ). Carbon starvation also reduces the analysis of gpa2 suggests that this gene product is in- cAMP level, even more sharply than nitrogen starvation volved in the regulation of the intracellular cAMP level (N. Mochizuki and M. Yamamoto, unpubl.). However, in accordance with nutritional conditions. Its most prob- excessive carbon starvation apparently shuts down any able function is modulation of adenylyl cyclase activity. developmental activities. Partial carbon starvation can Thus, it is likely that a GoL-subunit plays a role in trans- augment the induction of sexual development by nitro- duction of a signal from nutritional conditions, although gen starvation. It is thus conceivable that fission yeast the identity of the signal has still not been determined. has a mechanism that detects the availability of nutri- ents and regulates the level of intracellular cAMP. How cells monitor the availability of nutrition is an Results interesting but poorly understood question. It now ap- pears that our analysis of a G-protein homolog in S. Cloning of the gpa2 gene pombe has provided a clue to this question. S. pombe genomic DNA was analyzed by Southern blot- Heterotrimeric G proteins are generally coupled with ting using cDNAs encoding Dictyostelium discoideum Goal and Go~2 (Pupillo et al. 1989) as probes. A few re- striction fragments hybridized to these probes under low Present addresses: 1National Institute for Basic Biology, Okazaki 444, stringency conditions (data not shown), and we cloned Japan; 2Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA. an 8.0-kb EcoRI fragment that gave a clear hybridization 3Corresponding author. band with the G~I cDNA. Subcloning and partial se- GENES & DEVELOPMENT 6:2455-2462 91992 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/92 $3.00 2455 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Isshiki et al. quence analysis indicated that this fragment carried a -- 77:'7: :' ,r: ~7 :a;,~:~, :,V ;, ;,=,;,: ~V':':'V ;, :~:V:~ 7 ;,V:::: : :, .~T:x!V.~T;a.mVV,.~,;~::WWVV:,-'?,~::':Vr gene encoding a Got homolog, hereafter called gpa2. Be- :-" ;,at :,; 77.:7;,7: ; 7:7=, 7 :, r:T:7 vaq~';,.; 7A: ;: :~: :: ;,~ 7;:~.:,7/ "79; 7 A77 T :T,6,~,C;~ 7~::..-277T. ~. 4 2 ;,~,; "-T T .7.a~,~a 7 a3,aa=a,,=-'-a-TY;aaaT"7 : :ATCCava~A: 7 a'.;~*XT,:;;:;CT,~v,XZ:AAAzTT"TS:o(;TaTA;TTCA:;'2GA]Tq;A cause the 3' end of the gpa2 gene was not found on the 9 - 7.:~A7 ,KAT:~T'7~.'Z ,r : ;APT 7;7:" 7 ;TAAT,~.7 ~Z~.7 ;A A : a 7D:TT: qA, '7"YA,~:: ATA ".'WTG :',X T .~,AZ~': :,;,~,~; :~ 9 : :: ; :~-~: :;, :~ ;V:::;':: :V :'-. V;, ;,V ::aVVT~V: :: V;* a ;7~C':':,:~7V;,a,~<~,X,XT;k:~;,...":VT~..;, EcoRI fragment, we cloned a 2.4-kb PstI fragment that : ~ v : F x covers the missing part of the gene. A restriction map of ::a : :-:-::~:~ = :~= := : :,;: ::-: 7: ::-v ;rv ::-v ;,;.::,::7~:;:a:: 7:;:. :,;.-: .:a: ~,a,~.~,,~: ,,:;~,=..~ :,:7"r;/,>~.~:,a;,~,= the gpa2 locus is shown in Figure 1, and the nucleotide - : : sequence of the entire gpa2 open reading frame (ORF) : :4 ::>_a ::;.: :;::: '- 7 a_-,.; 7=:77~ :~a.~; .:_a;, : : :':a: ,=.:.~: .a ~A~:a7: ~C'JV~7~.a77.:: 3T;_Ta~22;TTr:TZTA VCTTt: :4 ::'a rr: 7"7 :,:7:" : ; r t,:7 *Xa;:7;,~ 7CC';A7;, e, :77 77 7"~;77:,; ),(77CT'STq 57.3TqP'~ AT;TT3Ta~TfiAGTT;72 ~ and its flanking regions was determined (see Fig. 2). This 424 7,~ : 7 77a 777 : 7 ;77: aYA: .k .~:AT:'7:~.'T. ;7"77:;:: ::,:~; A,~.aTT"7"D 77'_: 777;77"7: TTI~,,:,;,YT ;T :A~T~L sequence suggested the existence of a 405-bp-long intron in gpa2, which was confirmed by cDNA cloning using the polymerase chain reaction (PCR)method (Saiki et al. 1988) (data not shown). Many mammalian Got genes have an intron at the same position as gpa2 (Kaziro et al. 1991). The gpa2 gene has a coding capacity of 354 amino acids. The deduced Gpa2 protein exhibits 39.3% amino ...; ~,::7:;-:77 777 :a a.~ : .:.~ :,7 TC :27 ~ 7.'-7Ca 7: ~,~7~7 ~..A;~TA.Ka,~7"7 7:;CT,~A~,A;TAT7~ :JTkTA7 acid identity with D. discoideum Gotl, whose cDNA was used as the probe. Gpa2 is 40.7% identical with D. :4 :a~ a A=*-k 7 7:: : : 7 a:~: : 7t,::. ,~:7C 7.a,X; : 7"7:::_~ ;,;,: 77 :CC7~ :.77 UT'77APTC:'TT"T:TTC'TKAT,~7 discoideum Got2, 40.1% with human Gi2ot, and only 2'24 7 7 a:TZ'Tk?:a ~ aT :7 7:'Tk:AkATaCqZZ; A.aT&:;~:=, A:::; T; ::.A=,:" : ;GaA'7 k : ;A2C:::7 T :T.a3,:;A~.A..~:;A: : 24 aS : 77:'7 a :7 : V .~ .:~ ;:C a a':a7"P : TkT*Tk,~,~T,:,=: : :::,X~,:a: :=CL;,A:S'V,~Va A;T7 Ta:?';:-AVa, Taa.AVk:X":*7; 36.2% with S. pombe Gpal. It is most similar to Sac- :-24 :" :~777"77; ::,=?a: =a:':'7 ;-77:',%k:7-:777,= 7:':;,&a ;aT" :. : . ;;,;-7; ~'!*77;,A;,;7"Ta~U;,aV;, :':':: :" :W :;.~, ,~r,~;V" :-.4 7V~a :7C : A<: 7= :: 7 a .7~7 a-7 aA .~: :aT 7 : 977": ;. ;&;, "7;,7-7;: Va: & 77:4,T TG 7.~:77;T;~ :; ;7 ;7a: charomyces cerevisiae Gpa2 (42.4%) among G-protein ot-subunits identified so far (Fig. 3). Figure 2. The nucleotide and deduced amino acid sequences of gpa2. Numbering starts at the presumed initiation codon. Un- derlined regions match the consensus sequence for splicing in S. Disruption of the gpa2 gene pombe (Russell 1989). The region between a KpnI site and a ScaI site is shown, which has been sequenced in both directions. The One of the two gpa2 alleles in a diploid strain, JY765, nucleotide sequence data reported here have been submitted to was disrupted, as described in Materials and methods. the DNA Data Bank of Japan, EMBL, and GenBank nucleotide The structure of the disrupted allele, in which nucle- sequence data bases under accession number D13366. otides -22 to + 1514 in Figure 2 were deleted and re- placed by a ura4 + cassette, is schematically shown in Figure 1. Heterothallic haploid cells carrying the dis- rupted gpa2 allele were obtained by tetrad dissection of by genetic crosses. The homothallic derivatives mated the diploid strain. They were viable but were smaller in and sporulated readily in nitrogen-rich medium (Fig. 4B) cell size (Fig. 4E) and grew 1.4 times slower than the wild and grew very poorly. These phenotypes of the gpa2- type. Homothallic gpa2-disrupted cells were constructed disrupted cells were reminiscent of those of the cyrl- disrupted cells, which have no measurable activity of adenylyl cyclase and no measurable amount of cAMP (Maeda et al. 1990; Kawamukai et al. 1991). Addition of t t l lJ I ]; ,.I 2 mM cAMP and 5 mM caffeine to the medium, the latter of which is a phosphodiesterase inhibitor, could suppress pGPE1 3H I the observed phenotypes of the gpa2-disrupted cells: pGPR I Heterothallic cells resumed normal cell size and growth rate (Fig.
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